This application examines the role of the transmembrane domain in regulating several important receptor tyrosine kinases (RTKs), including Neu/ErbB2, FGFR3, KIT and RET. Mutations in each of these RTKS lead to significant human developmental and neoplastic syndromes. We have previously shown that a transmembrane domain consisting of the consensus repeat [VVVEVVV]n results in Neu/ErbB2 activation and that receptor activation is regulated by rotational coupling between the transmembrane and kinase domains. In Aim 1, using consensus sequence transmembrane domains to provide constitutive dimerization, we will examine rotational coupling for FGFR3, KIT and RET. Assays of receptor activation will include autophosphorylation, transformation and recruitment of effector proteins. Using chimeric RTKs, two biophysical approaches will also be employed: protein fragment complementation and fluorescence resonance energy transfer (FRET). In Aim 2, we will explore whether rotational coupling accompanies ligand-stimulated RTK activation. Using PDGFR-t3, chosen because it can be readily activated by its ligand, PDGF-BB, we will determine whether ligand binding to the extracellular domain induces rotational translation of the intracellular domain, coupled through the a-helical transmembrane domain. Receptor activation will be monitored by conventional assays such as autophosphorylation, as well as biophysical assays employing protein fragment complementation and FRET. These experiments will examine rotational coupling between the transmembrane domain and the kinase domain in response to ligandbinding. In Aim 3, we will examine the role of the juxtamembrane domain in regulating two different RTKs, RET and KIT. Mutations in the extracellular juxtamembrane domain of RET cause multiple endocrine neoplasia type 2A (MEN2A) and familial medullary thyroid carcinoma (FMTC), whereas mutations in the intracellular juxtamembrane domain of KIT lead to gastrointestinal stromal tumors (GIST) and mastocytoma. These experiments will thus investigate the mechanism whereby these gain-of-function mutations in the juxtamembrane domains of RET and KIT lead to receptor activation and downstream signal transduction.